Javascript is disabled by your browser. Some features of this Web site (e.g. Navigation menus, Text zoom, Hi-Contrast View, Quick Search, Random Imagery) will be impaired because of this. To navigate this Web site, please use the sitemap link on the top right corner of this page or the search facility on the left navigation bar. Alternatively, you could consider enabling javascript within your browser's settings.

Fluorescence lifetime based pH sensing

Objectives:

This project aims to develop high accuracy optical pH sensors suitable for biological and clinical applications. We are using fluorescence lifetime methods because they offer a convenient route to the development of minimally invasive monitoring methods. The fluorescence lifetime is the average time that a molecule spends in the excited state before emitting a photon of light and returning to the ground state; most biological molecules have lifetimes in the picosecond (10
-12 sec) to microsecond (10
-6 sec) range. Fluorescence lifetime measurements are independent of signal level and have been used to obtain pH measurements from cellular material and to measure oxygen content through skin [
1,2]. This is a significant advantage compared to optical sensors that rely on fluorescence intensity and/or wavelength measurements, which can be adversely affected by light scattering/absorption effects due to tissue inhomogenity.

Our pH sensors are based on relatively short-lived species with nanosecond lifetimes, and the primary fluorophore is acridine. This molecule exhibits a large difference in lifetime between the protonated and base forms making it a candidate for pH sensing. There are however a number of difficulties such as excited state protonation and halide quenching to be overcome before acridine can be used as a pH sensor [
3,4]. We have also examined resorufin as a possible lifetime based pH sensor using 460 nm LED excitation [
5].

Acridine in 0.1 M phosphate buffer:

Acridine is a pH sensitive dye that exhibits a large difference in fluorescence lifetime, between the neutral and protonated states. The plot on the left shows the difference in the fluorescence decay curves (380 nm excitation) for acridine in 0.1 M phosphate buffer at two different pH values. It is clear that each decay curve comprises of two emitting species, and that the proportion of each changes with pH.
The protonated species has a lifetime of ~31.6 ns in 1M HNO3 while the neutral base form has a lifetime of 6.6 ns in 1 M NaOH.

If we plot the change in the measured average lifetime versus pH at two different emission wavelengths we get the plots on the right. The relationship between pH and average lifetime fits to a simple polynomial over the physiologically important pH 5.8 - 8.1 range.
The average lifetime is longer at longer emission wavelengths because the protonated form of acridine emits at longer wavelength than the neutral form.

Unfortunately acridine fluorescence can be quenched by the presence of species such as halide ions. The plots opposite show the difference in average lifetime (measured at 450 nm) for acridine (5 x 10-3 M) in solution when NaCl is added. The dramatic drop in the average lifetime is due to the dynamic quenching of the protonated species.
This along with the fact that acridine undergoes an excited state protonation makes acridine unsuitable as a pH sensor except under very well controlled circumstances.

Our next step was therefore to find some way of negating the halide quenching and excited state effects, and to do this we deployed a protective support material.

Protected Acridine:

We have incorporated acridine into a transparent, anion exclusion membrane in order to prevent halide quenching. Preliminary results are shown on the right.
For both 450 and 500 nm emission wavelengths the average lifetime is seen to vary over the 8-11 pH unit range. Furthermore, the unique membrane excludes anionic quenchers such as halides, while still allowing free transport of hydrogen ions.

The shift in ground state pKa is due to the unique environments that acridine experiances in the membrane. This large shift (~ 4 pH units) in the ground state pKa ensures that ground state protonation is not an issue any more. [
note the pKa values for solution and membrane acridine are dependant on the emission wavelength sampled]